This invention relates to combinatorial libraries and methods for their generation.
The rapid production of diverse collections or libraries of chemical compounds is an important goal for those desiring to screen large numbers of novel compounds or diversomers for pharmacological activity. Combinatorial synthesis has been utilized to create libraries of molecules. These libraries often consist of oligomeric or polymeric molecules created from the sequential addition of monomeric subunits. However, typically the monomer subunits utilized have been amino acids, nucleic acid bases or carbohydrates. The reactions used to couple the subunits are standard reactions such as dehydration synthesis reactions.
The initial report of rapid concurrent solid phase synthesis by Geysen and co-workers, Geysen, H. M.; Meleon, R. H.; Barteling, S. J., Proc. Natl. Acad. Sci. USA, Vol. 81, p. 3998 (1984), described the construction of multi-amino acid peptide libraries. Houghten et al., 354 Nature 84, 1991 and WO 92/09300 (PCT/US91/08694), describe the generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. These libraries are composed of mixtures of free peptides which form a heterogenous library. Systematic identification of optimal peptide ligands is achieved by screening a library followed by iterative selection and synthesis processes. For example, one library consisted of a series of six residue peptides having the first two positions specifically defined, and the last four positions consisting of a random mixture of 18 L-amino acids. This library was screened to determine which pair of defined peptides had optimum activity in an assay. A second library was then synthesized in which the optimal pair of peptides were included, the third position of each peptide individually synthesized, and the last three peptides consisted of a random mixture of 18 L-amino acids. This library was screened as before and the process repeated until the optimum six residue peptide was identified. Houghten et al. state:
"A number of other libraries, such as one composed entirely of D-amino acids, have been prepared which in total permit the systematic screening of hundreds of millions of peptides. PA1 "It is clearly not enough to use a random mixture of activated amino acids in a peptide synthesis protocol, because the widely different coupling rates of different amino acids will lead to unequal representation and because each bead will contain a mixture of different peptides. Our solution was to use a `split synthesis` approach. The first cycle consisted of distributing a pool of resin beads into separate reaction vessels each with a single amino acid, allowing the coupling reactions to go to completion, and then repooling the beads. The cycle was repeated several times to extend the peptide chain. In this fashion, each bead should contain only a single peptide species." The library of beads was screened by a staining procedure and stained beads visualized using a microscope, and removed. The structure of the peptide is obtained by a chemical analysis of the material on the single bead. Lam et al. indicate: PA1 "Additionally, our approach has far greater potential for applying the richness of well-established peptide chemistry to synthesize libraries incorporated D-amino acids or unnatural amino acids as well as specific secondary structures including cyclic peptides. All of this can be accomplished without need to keep records of the synthetic products as our interest is focused just on those peptides which provide a strong interaction signal with the acceptor."
A fundamental feature of SPCLs synthetic peptide combinatorial libraries! is that free peptides can be generated and used in solution in virtually all existing assay systems at a concentration of each peptide most applicable to the assay. This approach has also been successfully used in radio-receptor assays (opioid peptides) and plaque inhibition assays (human immunodeficiency virus (HIV-1) and Herpes Simplex Virus (HSV)). SPCLs, as described, greatly aid all areas of drug discovery and research involving peptides."
Lam et al., 354 Nature 82, 1991, and WO 92/00091 (PCT/US91/04666) and Houghten et al., 354 Nature 84, 1991 and WO 92/09300 (PCT/US91/08694), herein, describe systematic synthesis and screening of peptide and other libraries of defined structure. The method used is based on a one bead one peptide approach in which a large peptide library consisting of millions of beads are screened. Each bead contains a single peptide. The authors state:
Dower et al., WO 91/19818 (PCT/US91/04384) describes peptide libraries expressed as fusion proteins of bacteriophage coat proteins.
Dower et al., WO 93/06121 (PCT/US92/07815) describes a method for synthesizing random oligomers and the use of identification tags to identify oligomers with desired properties.
Ellman, U.S. Pat. No. 5,288,514 describes the solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support.
Huebner, U.S. Pat. No. 5,182,366 describes the controlled synthesis of peptide mixtures using mixed resins.
Several groups have focused on a combinatorial approach for the construction of peptide libraries in order to randomly screen for drug candidates. However, although a viable strategy for the identification of potential new compounds for medicinal chemists, the standard gamut of problems associated with peptide based drugs i.e. poor bio-availability, etc. must still be addressed. Combinatorial synthesis has recently been adapted for the assembly of nucleic acid, carbohydrate, and even benzodiazepine libraries.
The continued development of strategies for labelling and/or tagging materials within the libraries has greatly expedited and simplified the process of identification of active constituents within groups of diversomers. Once a potentially useful compound has been characterized from a peptide library, medicinal chemists must shift to peptidomimetic synthesis in order to convert these potentially promising peptide compounds into small organic molecules. Hence, devising a combinatorial strategy incorporating peptidomimetics could greatly simplify the drug discovery process by providing more useful libraries of small organic molecules, and may itself directly produce a drug, an unlikely accomplishment within peptide libraries.